Vascular oxidative stress is a key component of many lung diseases, but its treatment is inadequate, in part due to sub-optimal delivery of antioxidants to endothelial cells (EC). Previous studies have shown that: 1) antibodies to Platelet Endothelial Adhesion Molecule (PECAM) permit intracellular delivery of drugs to EC via a novel endocytotic pathway distinct from clathrin- and caveoli-mediated endocytosis; and 2) the antioxidant enzyme (AOE) catalase conjugated with anti-PECAM accumulates in the lungs after IV injection and protects against oxidative lung injury in some animal models. The aim of this translational grant is to enhance the duration and effectiveness of protective effects of this promising strategy to clinically significant levels. We hypothesize that: i) the activity of the conjugates can be prolonged by optimization of their design and by manipulating intracellular trafficking and lysosomal degradation; ii) targeted delivery of additional AOEs to EC will permit enhanced protection from oxidative stress; and iii) pulmonary targeting of optimized AOE conjugates will be protective against hyperoxia. We will test this in the following Specific Aims: 1) define the mechanisms of metabolism of anti-PECAM conjugates in EC. We will test the hypothesis that endocytosis and trafficking involve the cytosolic domain of PECAM, Na+-H+ exchangers (NHE) and rearrangements of cytoskeleton, which can be affected by auxiliary agents to prolong duration of conjugates; 2) design targeting of additional AOE. To enhance protection, tandem SOD/catalase (to detoxify 02- and H202) and 1-CysPrx peroxiredoxin (to detoxify H202 and lipid peroxides) conjugates will be produced. Their composition, activities, EC uptake and protection will be studied in cell culture, while their pharmacokinetics and pulmonary targeting will be determined in naive animals and animals with oxidative lung injury; and 3) evaluate protective effects of the conjugates in animal models. The effectiveness and duration of the effects of new conjugates will be compared and regimens of AOE targeting and mechanisms of protection will be studied in a model of acute EC injury induced by H202 in the pulmonary vasculature in mice. Finally, protection against hyperoxic lung injury by best conjugates will be tested. The overall goal of this proposal is to optimize strategies for vascular AOE targeting, to ultimately initiate translation of this strategy into the clinical domain.